Lsh is a member of the SNF2 family of chromatin remodelers, that regulate diverse biological processes such as replication, repair and transcription. Although expression of Lsh is highly tissue specific in adult animals, Lsh mRNA is detectable in multiple tissues during embryogenesis. In order to determine the physiologic role of Lsh during murine development and to assess its unique function in adult mice, we performed targeted deletion of the Lsh gene using homologous recombination in murine embryonic stem cells. Lsh-/- embryos occurred with the expected Mendelian frequency after implantation and during embryogenesis. However, Lsh-/- mice died within a few hours after birth. Furthermore, newborn mice were 22% lower in weight in comparison with their littermates and showed renal lesions. Thus Lsh is a non-redundant member of the SNF2 family and is essential for normal murine development and survival.

The function of MADS-box genes in flower and fruit development has been uncovered at a rapid pace over the past decade. Evolutionary biologists can now analyse the expression pattern of MADS-box genes during the development of different plant species, and study the homology of body parts and the evolution of body plans. These studies have shown that floral development is conserved among divergent species, and indicate that the basic mechanism of floral patterning might have evolved in an ancient flowering plant.

Members of the T-box gene family have been identified in both vertebrates and invertebrates, where they play key roles in the regulation of embryonic development, and particularly in morphogenesis and the assignment of cell fate. T-box proteins act as transcription factors which regulate the expression of downstream effector genes. This review focuses on the identification of T-box target genes and the basis of T-box functional specificity.

Canine homolog of the T-box transcription factor T; failure of the protein to bind to its DNA target leads to a short-tail phenotype.

Mamm Genome. 2001; 12: 212-8

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Domestic dog breeds show a wide variety of morphologies and offer excellent opportunities to study the molecular genetics of phenotypic traits. We are interested in exploring this potential and have begun by investigating the genetic basis of a short-tail trait. Our focus has been on the T gene, which encodes a T-box transcription factor important for normal posterior mesoderm development. Haploinsufficiency of T protein underlies a short-tail phenotype in mice that is inherited in an autosomal dominant fashion. We have cloned the dog homolog of T and mapped the locus to canine Chromosome (Chr) 1q23. Full sequence analysis of the T gene from a number of different dog breeds identified several polymorphisms and a unique missense mutation in a bob-tailed dog and its bob-tailed descendants. This mutation is situated in a highly conserved region of the T-box domain and alters the ability of the T protein to bind to its consensus DNA target. Analysis of offspring from several independent bobtail x bobtail crosses indicates that the homozygous phenotype is embryonic lethal.

The T-box gene family was uncovered less than a decade ago but has been recognized as important in controlling many and varied aspects of development in metazoans from hydra to humans. Extensive screening and database searching has revealed several subfamilies of genes with orthologs in species as diverse as Caenorhabditis elegans and humans. The defining feature of the family is a conserved sequence coding for a DNA-binding motif known as the T-box, named after the first-discovered T-box gene, T or Brachyury. Although several T-box proteins have been shown to function as transcriptional regulators, to date only a handful of downstream target genes have been discovered. Similarly, little is known about regulation of the T-box genes themselves. Although not limited to the embryo, expression of T-box genes is characteristically seen in dynamic and highly specific patterns in many tissues and organs during embryogenesis and organogenesis. The essential role of several T-box genes has been demonstrated by the developmental phenotypes of mutant animals.

Targeted disruption of the CP2 gene, a member of the NTF family of transcription factors.

J Biol Chem. 2001; 276: 7836-42

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The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse as Drosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, we have generated mice nullizygous for the CP2 allele. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the -/- lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homologue of LBP-1a, was observed with these extracts. Furthermore, we demonstrate that recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene.

T-box genes encode a family of phylogenetically conserved DNA-binding proteins that regulate gene expression during embryogenesis. While the developmental importance of many T-box genes has been well documented, little is known about how family members differ in their DNA binding properties and ability to modulate transcription. Here we show that although TBX1, TBX2 and the Xenopus T protein (Xbra) share only 50-60% identity within their DNA-binding domains they can bind the same DNA sequence in vitro. However, the proteins differ in three important respects. While TBX1 protein binds a palindromic T oligonucleotide as a dimer, as had been previously reported for Xbra, TBX2 appears to bind the same DNA sequence as a monomer. Also, T protein/DNA complexes are stabilized in vitro by the addition of specific antibodies, whereas TBX2/DNA complexes are not stabilized by antibodies. Most importantly, TBX2 represses while Xbra activates transcription of the same chimeric reporter plasmid. TBX1, although capable of binding to the chimeric promoter, has no effect on transcription. Thus, while the DNA binding domains of T-box proteins share substantial homology, these proteins differ in both their DNA binding and transcriptional modulation properties. These results suggest that the various T-box proteins, while highly conserved, likely use different mechanisms to modulate transcription and may have different targets in vivo.

Six family genes--structure and function as transcription factors and their roles in development.

Bioessays. 2000; 22: 616-26

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The members of the Six gene family were identified as homologues of Drosophila sine oculis which is essential for compound-eye formation. The Six proteins are characterized by the Six domain and the Six-type homeodomain, both of which are essential for specific DNA binding and for cooperative interactions with Eya proteins. Mammals possess six Six genes which can be subdivided into three subclasses, and mutations of Six genes have been identified in human genetic disorders. Characterization of Six genes from various animal phyla revealed the antiquity of this gene family and roles of its members in several different developmental contexts. Some members retain conserved roles as components of the Pax-Six-Eya-Dach regulatory network, which may have been established in the common ancestor of all bilaterians as a toolbox controlling cell proliferation and cell movement during embryogenesis. Gene duplications and cis-regulatory changes may have provided a basis for diverse functions of Six genes in different animal lineages.

Mapping and developmental expression analysis of the WD-repeat gene Preb.

Genomics. 2000; 63: 391-9

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We have isolated from mouse a novel WD-motif-containing gene designated Preb. This gene encodes a predicted protein of 416 amino acids and has significant homology with other members of the WD-motif gene superfamily that play a role in cell fate determination. Preb maps to the proximal end of chromosome 5 in mouse, near the Hmx1 homeobox gene. Preb is detectable in early stage embryos in the peripheral nervous system, developing liver, and surface ectoderm. Later, Preb is expressed in the anterior portion of Rathke's pouch, which gives rise to the anterior pituitary, the organ responsible for the production of prolactin and other hormones. In midgestation embryos, the most extensive expression of Preb is observed in the perichondrium of the craniofacial, axial, and appendicular skeleton. The expression pattern of Preb in murine embryos suggests a potential role in the specification of multiple cell types, in particular, the fetal skeleton.

The mammalian Pax gene family comprises nine members that are characterized by a conserved DNA-binding motif, the paired domain, which was originally described in the Drosophila protein paired. Both loss- and gain-of-function studies reveal that Pax genes carry out essential roles during embryogenesis, and in some instances, may function as master regulatory genes. This review focuses on both genetic and biochemical aspects of the Pax family, and emphasizes important differences in the activity of individual Pax genes and their protein products.

The lin-31 gene is required for the proper specification of vulval cell fates in the nematode Caenorhabditis elegans and encodes a member of the winged-helix family of transcription factors. Members of this important family have been identified in many organisms and are known to bind specific DNA targets involved in a variety of developmental processes. DNA sequencing of 13 lin-31 alleles revealed six nonsense mutations and two missense mutations within the DNA-binding domain, plus three deletions, one transposon insertion, and one frameshift mutation that all cause large-scale disruptions in the gene. The missense mutations are amino acid substitutions in the DNA-binding domain and probably disrupt interactions of the LIN-31 transcription factor with its DNA target. In addition, detailed phenotypic analysis of all 19 alleles showed similar penetrance for several characteristics examined. From our analysis we conclude: (1) the null phenotype of lin-31 is the phenotype displayed by almost all of the existing alleles, (2) the DNA-binding domain plays a critical role in LIN-31 function, and (3) direct screens for multivulva and vulvaless mutants will probably yield only null (or strong) alleles of lin-31.

Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors.

Gene. 2000; 246: 285-93

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The RFX family of transcription factors is characterized by a unique DNA binding domain. Five genes have been isolated in mammals, one gene in Caenorhabditis elegans and in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Whereas the roles of the RFX genes are beginning to be understood in yeasts, no clear function has been reported in multicellular organisms, except for RFX5, the most divergent member of the family. To study the physiological role of RFX transcription factors using an alternative multicellular model, we report the isolation and characterization of the Drosophila RFX gene (dRFX). The fruit fly protein shares highly conserved domains with the mammalian factors RFX1 to 3 and is more closely related to this subgroup. It binds DNA with the same target specificity as mammalian factors RFX1 to 3. dRFX is located on chromosome III and we characterized the entire locus. dRFX expression was analyzed during embryogenesis. dRFX mRNAs are detected only in the peripheral nervous system and in the brain of the embryo.

The Nuclear Factor I (NFI) family of site-specific DNA-binding proteins (also known as CTF or CAAT box transcription factor) functions both in viral DNA replication and in the regulation of gene expression. The classes of genes whose expression is modulated by NFI include those that are ubiquitously expressed, as well as those that are hormonally, nutritionally, and developmentally regulated. The NFI family is composed of four members in vertebrates (NFI-A, NFI-B, NFI-C and NFI-X), and the four NFI genes are expressed in unique, but overlapping, patterns during mouse embryogenesis and in the adult. Transcripts of each NFI gene are differentially spliced, yielding as many as nine distinct proteins from a single gene. Products of the four NFI genes differ in their abilities to either activate or repress transcription, likely through fundamentally different mechanisms. Here, we will review the properties of the NFI genes and proteins and their known functions in gene expression and development.

Classical mutations at the mouse Brachyury (T) locus were discovered because they lead to shortened tails in heterozygous newborns. no tail (ntl) mutants in the zebrafish, as their name suggests, show a similar phenotype. In Drosophila, mutants in the brachyenteron (byn) gene disrupt hindgut formation. These genes all encode T-box proteins, a class of sequence-specific DNA binding proteins and transcription factors. Mutations in the C. elegans mab-9 gene cause massive defects in the male tail because of failed fate decisions in two tail progenitor cells. In a recent paper, Woollard and Hodgkin have cloned the mab-9 gene and found that it too encodes a T-box protein, similar to Brachyury in vertebrates and brachyenteron in Drosophila. The authors suggest that their results support models for an evolutionarily ancient role for these genes in hindgut formation. We will discuss this proposal and try to decide whether the gene sequences, gene interactions and gene expression patterns allow any conclusions to be made about the rear end of the ancestral metazoan.

Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

We have identified and characterized a new member of the mammalian brain-specific T-box gene family, Tbr2, which is closely related to mouse Tbr1, and to the Xenopus earliest mesodermal gene, Eomesodermin. As Tbr1, Tbr2 is predominantly expressed in some regions of the developing brain, but in a strikingly complementary manner. On embryonic day 14.5 (E14.5), Tbr2 mRNA expression was observed in the mesencephalon and rhombencephalon in contrast to Tbr1 which was expressed mostly in the telencephalon. At this stage, Tbr2 mRNA was readily detectable in the postmitotic and differentiating neurons located in various brain regions, i.e., oculomotor, red, trigeminal, vestibular, facial, and hypoglossal nuclei. However, expression of Tbr2 in these nuclei became undetectable on E18.5. In contrast, Tbr2 mRNA expression was detected in the hippocampus only from E18.5 onwards. Whereas Tbr2 expression disappeared in most parts of the mature adult brain, it remained detectable in the hippocampus and olfactory bulb, regions where some neuronal precursors retain their differentiation potential. These results suggest that Tbr2 may play a crucial role in differentiating neurons rather than in proliferating or already differentiated neurons. In addition, similarly to Xenopus Eomesodermin, mouse Tbr2 showed biphasic expression; a first peak around E6.5 and a second peak around E14.5, suggesting that Tbr2 may also be important at early stages of gastrulation.

Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development.

Mech Dev. 1999; 84: 133-8

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The members of the T-box gene family share a highly conserved DNA binding domain named the T-domain, and important developmental functions. Here we report the cloning of chicken Tbr1 and of murine and chicken Tbr2 (orthologs of the Xenopus eomesodermin gene), the mapping of the murine Tbr2 to chromosome 9, and their pattern of expression during mouse and chick embryogenesis. Both Tbr 1 and 2 have a restricted and conserved domain of expression in the telencephalic pallium of the two species. Chick Tbr2 has a specific and dynamic expression in the gastrulating embryo.

Mga, a dual-specificity transcription factor that interacts with Max and contains a T-domain DNA-binding motif.

EMBO J. 1999; 18: 7019-28

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The basic-helix-loop-helix-leucine zipper (bHLHZip) proteins Myc, Mad and Mnt are part of a transcription activation/repression system involved in the regulation of cell proliferation. The function of these proteins as transcription factors is mediated by heterodimerization with the small bHLHZip protein Max, which is required for their specific DNA binding to E-box sequences. We have identified a novel Max-interacting protein, Mga, which contains a Myc-like bHLHZip motif, but otherwise shows no relationship with Myc or other Max-interacting proteins. Like Myc, Mad and Mnt proteins, Mga requires heterodimerization with Max for binding to the preferred Myc-Max-binding site CACGTG. In addition to the bHLHZip domain, Mga contains a second DNA-binding domain: the T-box or T-domain. The T-domain is a highly conserved DNA-binding motif originally defined in Brachyury and characteristic of the Tbx family of transcription factors. Mga binds the preferred Brachyury-binding sequence and represses transcription of reporter genes containing promoter-proximal Brachyury-binding sites. Surprisingly, Mga is converted to a transcription activator of both Myc-Max and Brachyury site-containing reporters in a Max-dependent manner. Our results suggest that Mga functions as a dual-specificity transcription factor that regulates the expression of both Max-network and T-box family target genes.

Brachyury is the founder member of the T-box family of transcription factors, which is characterized by a DNA-binding domain of approximately 200 amino acids. Members of the T-box gene family play important roles in the development of both vertebrate and invertebrate embryos, including the control of gastrulation, development of the heart, and perhaps even the decision as to whether to form arm or leg. An understanding of how the T-box genes act requires analysis of how their expression is controlled, identification of their target genes, and an insight into how different family members exert different effects.

The role of mammalian Hox genes in regulating segmental patterning of axial structures and the limb is well established. A similar role in development of soft tissue organ systems has recently been suggested by observations linking several 5' members of the HoxA and HoxD clusters to segmentation events and morphogenesis in the gastrointestinal and genitourinary systems. We have specifically examined the role of Hoxa-10 in development of the male accessory sex organs by characterizing expression of Hoxa-10 in the developing male reproductive tract and correlating expression to morphologic abnormalities in knockout mice deficient for Hoxa-10 function. We report that Hoxa-10 expression in the Wolffian duct and urogenital sinus is regionally restricted and temporally regulated. The domain of expression is defined anteriorly by the caudal epididymis and extends posteriorly to the prostatic anlagen of the urogenital sinus. Expression was maximal at E18 and down-regulated postnatally, well before accessory sex organ morphogenesis is completed. Expression in the prostatic anlagen of the urogenital sinus cultured in vitro does not depend upon the presence of testosterone. Loss of Hoxa-10 function is associated with diminished stromal clefting of the seminal vesicles and decreased size and branching of the coagulating gland. The ductal architecture of the coagulating gland was altered in approximately 30% of mutants examined and suggests a partial posterior morphologic transformation of the coagulating gland. We interpret these data to indicate that Hoxa-10 is expressed in a region specific manner during late gestation and into the perinatal period and that Hoxa-10 is required for normal accessory sex organ development.

Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis.

Mech Dev. 1999; 80: 219-21

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T-box genes are conserved in all animal species. We have identified two members of the T-box gene family from the zebrafish, Danio rerio. Zf-tbr1 and zf-tbx3 share high amino acid identity with human, murine, chick and Xenopus orthologs and are expressed in specific regions during zebrafish development.

Several genes containing the conserved T-box region in invertebrates and vertebrates have been reported recently. Here, we describe three novel members of the T-box gene family in zebrafish. One of these genes, tbx-c, is studied in detail. It is expressed in the axial mesoderm, notably, in the notochordal precursor cells immediately before formation of the notochord and in the chordoneural hinge of the tail bud, after the notochord is formed. In addition, its expression is detected in the ventral forebrain, sensory neurons, fin buds and excretory system. The expression pattern of tbx-c differs from that of the other two related genes, tbx-a and tbx-b. The developmental role of tbx-c has been analysed by overexpression of the full-length tbx-c mRNA and a truncated form of tbx-c mRNA, which encodes the dominant-negative Tbx-c. Overexpression of tbx-c causes expansion of the midline mesoderm and formation of ectopic midline structures at the expense of lateral mesodermal cells. In dominant-negative experiments, the midline mesoderm is reduced with the expansion of lateral mesoderm to the midline. These results suggest that tbx-c plays a role in formation of the midline mesoderm, particularly, the notochord. Moreover, modulation of tbx-c activity alters the development of primary motor neurons. Results of in vitro analysis in zebrafish animal caps suggest that tbx-c acts downstream of early mesodermal inducers (activin and ntl) and reveal an autoregulatory feedback loop between ntl and tbx-c. These data and analysis of midline (ntl-/- and flh-/-) and lateral mesoderm (spt-/-) mutants suggest that tbx-c may function during formation of the notochord.

Expression of five novel T-box genes and brachyury during embryogenesis, and in developing and regenerating limbs and tails of newts.

Dev Growth Differ. 1999; 41: 321-33

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Several T-box genes are considered to play important roles in developing limbs, tails and neural retinae. Five novel T-box genes in the Japanese newt were isolated and their expression was analyzed, together with another T-box gene of brachyury, during embryogenesis and in the developing and regenerating limbs and tail. Four are designated CpTbx2, CpTbx3, CpTbx6R and CpEomesodermin based on molecular phylogenetic analyses, and the other is named CpUbiqT from its ubiquitous expression. While all were expressed during embryogenesis, only four of them (CpTbx2, CpTbx3, CpUbiqT and brachyury) were detected in developing limbs and/or tails. Except for brachyury, they were continuously expressed in normal adult appendages and showed elevated expression levels in regenerating limbs, whereas only CpTbx2 showed significant up-regulation in regenerating tails. Compared with orthologous genes in other species, CpTbx2, CpTbx3 and CpEomesodermin showed several notable differences such as an abundance of maternal transcripts of CpEomesodermin, a unique insertion sequence within the T-box domain of CpTbx2, and a lack of visible expression of CpTbx2and CpTbx3 in the apical ectodermal region of developing limbs. In view of the uniqueness of the newt, these results are discussed with respect to the possibility of their involvement in regeneration.

Characterization of the zebrafish tbx16 gene and evolution of the vertebrate T-box family.

Dev Genes Evol. 1998; 208: 94-9

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We report on a new zebrafish T-box-containing gene, tbx16. It encodes a message that is first detected throughout the blastoderm soon after the initiation of zygotic gene expression. Following gastrulation, expression becomes restricted to paraxial mesoderm and later primarily to the developing tail bud. To gain an evolutionary prospective on the potential function of this gene, we have analyzed its phylogenetic relationships to known T-box genes from other species. Zebrafish tbx16 is likely orthologous to the chicken Tbx6L and Xenopus Xombi/Antipodean/Brat/VegT genes. Our analysis also shows that zebrafish tbx6 and mouse Tbx6 genes are paralogous to zebrafish tbx16. We present evidence which argues, that despite the same name and similar expression, zebrafish tbx6 and mouse Tbx6 genes are not orthologous to each other but instead represent relatively distant paralogs. The expression patterns of all genes are discussed in the light of their evolutionary relationships.

We have identified a cDNA encoding a novel Xenopus winged helix transcription factor termed as XFD-11 (Xenopus fork head domain). The DNA binding domain is most closely related to those of human or murine FREAC-3 (FKHL7/MF-1/FKH-1) proteins. The XFD-11 gene is activated at late blastula/early gastrula and transcription proceeds throughout embryogenesis. Early expression is found in ventral and lateral but not in dorsal mesoderm. At neurula stages, transcripts are found in posterior mesoderm except for the dorsal midline, and the gene is also transcribed at the lateral border of the neural plate and within anterior neuroectoderm. At later stages of development, transcripts are detected within the pronephros, the heart, within neural crest cells surrounding the eye, in the mandibular, hyoid and branchial arches, and within the tail.

AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice.

Proc Natl Acad Sci U S A. 1998; 95: 13714-9

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The homozygous disruption of the mouse AP-2 gene yields a complex and lethal phenotype that results from defective development of the neural tube, head, and body wall. The severe and pleiotropic developmental abnormalities observed in the knockout mouse suggested that AP-2 may regulate several morphogenic pathways. To uncouple the individual developmental mechanisms that are dependent on AP-2, we have now analyzed chimeric mice composed of both wild-type and AP-2-null cells. The phenotypes obtained from these chimeras indicate that there is an independent requirement for AP-2 in the formation of the neural tube, body wall, and craniofacial skeleton. In addition, these studies reveal that AP-2 exerts a major influence on eye formation, which is a critical new role for AP-2 that was masked previously in the knockout mice. Furthermore, we also have uncovered an unexpected influence of AP-2 on limb pattern formation; this influence is typified by major limb duplications. The range of phenotypes observed in the chimeras displays a significant overlap with those caused by teratogenic levels of retinoic acid, strongly suggesting that AP-2 is an important component of the mechanism of action of this morphogen.

Cloning, mapping, and expression analysis of TBX15, a new member of the T-Box gene family.

Genomics. 1998; 51: 68-75

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The T-box gene family has been conserved throughout metazoan evolution and codes for putative transcription factors that share a uniquely defining DNA-binding domain. We have previously uncovered six mouse T-box genes with discrete spatial and temporal patterns of expression during embryogenesis. Here, we report a novel mouse T-box gene, Tbx15. The Tbx15 gene produces a 3.7-kb transcript with an open reading frame coding for a polypeptide with 602 amino acid residues. Phylogenetic analysis places the Tbx15 gene into a T-box subfamily that also includes mouse Tbx1, Drosophila H15, and nematode Ce-tbx-12 genes. We have mapped mouse Tbx15 to chromosome 3, at a position 49 cM from the centromere. During development, Tbx15 transcripts are first detected at embryonic day 9.5. The gene is expressed primarily in the cranio-facial region and in the developing limbs. An isolated human homolog, TBX15, has been mapped by in situ hybridization to chromosomal band 1p13. TBX15 appears to be an excellent candidate for the dominantly expressed acromegaloid facial appearance syndrome, which also maps to the short arm of human chromosome 1 and, like TBX15, is expressed prominently in the eyebrow regions.

The transcription factor Oct-4 is expressed specifically in the totipotent germline cycle of mice. Cells that lose Oct-4 differentiate along different paths to form embryonic and extraembryonic somatic tissue. Oct-4 may maintain the potency of stem and germline cells by preventing all other differentiation pathways. Oct-4 may also regulate the molecular differentiation of cells in the germ lineage as it progresses from the fertilized egg, through cleavage stage/morula blastomeres, blastocyst, inner cell mass, epiblast, germ cells, and gametes. The factors that regulate, and are regulated by, Oct-4 are reviewed with respect to the phenomena of cell potency and germ/soma segregation and differentiation.

A combined analysis of genomic and primary protein structure defines the phylogenetic relationship of new members if the T-box family.

Genomics. 1998; 48: 24-33

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T-box genes form an ancient family of putative transcriptional regulators characterized by a region of homology to the DNA-binding domain of the murine Brachyury (T) gene product. This T-box domain is conserved from Caenorhabditis elegans to human, and mutations in T-box genes have been associated with developmental defects in Drosophila, zebrafish, mice, and humans. Here we report the identification of three novel murine T-box genes and an investigation of their evolutionary relationship to previously known family members by studying the genomic structure of the T-box. All T-box genes from nematodes to humans possess a characteristic central intron that presumably was inherited from a common ancestral precursor. Two additional intron positions are also conserved with the exception of two nematode T-box genes. Subsequent intron insertions, potential deletions, and/or intron sliding formed a structural basis for the divergence into distinct subfamilies and a substrate for length variations of the T-box domain. In mice, the 11 T-box genes known to date can be grouped into seven subfamilies. Genes assigned to the same subfamily by genomic structure show related expression patterns. We propose a model for the phylogenetic relationships within the gene family that provides a rationale for classifying new T-box genes and facilitates interspecific comparisons.

TBX2 is a member of a recently discovered gene family of transcription factors, named T-box genes after the Brachyury or T gene. Mutations in two of these family members, TBX5 and TBX3, have recently been shown to be responsible for the congenital abnormalities associated with Holt Oram syndrome and ulnar-mammary syndrome respectively, while mutations in T-box genes in other species also result in developmental abnormalities in the tissues where the gene is normally expressed. Thus, it likely that other T-box genes are responsible for additional human developmental anomalies. Here we report the exon/intron boundaries of TBX2 and a polymorphism within intron 2 of TBX2 that should be useful for exploring the involvement of this gene in human genetic disease. We further note that the exon/intron boundaries of TBX2 are highly conserved within the T-box domain with those of both T and TBX5, as well as with a new human T-box gene and more distantly related genes from Caenorhabditis elegans and Drosophila. This observation should facilitate the analysis of the genomic structure of other members of this gene family. It is also of interest that several members of this gene family have an additional intron that is variably present within members of at least two different lineages of the T-box family. This observation has implications regarding the evolution of T-box genes.

T-box genes, in all metazoans studied from nematode to man, exist in small gene families. They encode transcription factors with a novel, large, and highly conserved DNA binding domain termed the T-domain. In all cases studied, T-box genes have important developmental roles. Two familial diseases, Holt-Oram syndrome and ulnar-mammary syndrome, were recently shown to be caused by mutations in the human T-box genes TBX5 and TBX3, respectively. T-box genes were first identified in Drosophila and mouse. Two of the three known Drosophila T-box genes show a close sequence homology to mammalian genes. Similarities in the phenotypes of fly and mammalian mutants can be taken as evidence of functional conservation. We report here the isolation of a fourth Drosophila T-box gene, optomotor-blind-related gene-1 (org-1), closely related to mouse and human TBX1. We localized TBX1 to chromosomal band 22q11, confirming a recent report, and discuss TBX1 as a candidate gene for DiGeorge and related syndromes.

Inhibition of fibroblast growth factor (FGF) signaling prevents trunk and tail formation in Xenopus and zebrafish embryos. While the T-box transcription factor Brachyury (called No Tail in zebrafish) is a key mediator of FGF signaling in the notochord and tail, the pathways activated by FGF in non-notochordal trunk mesoderm have been uncertain. Previous studies have shown that the spadetail gene is required for non-notochordal trunk mesoderm formation; spadetail mutant embryos have major trunk mesoderm deficiencies, but relatively normal tail and notochord development. We demonstrate here that spadetail encodes a T-box transcription factor with homologues in Xenopus and chick. Spadetail is likely to be a key mediator of FGF signaling in trunk non-notochordal mesoderm, since spadetail expression is regulated by FGF signaling. Trunk and tail development are therefore dependent upon the complementary actions of two T-box genes, spadetail and no tail. We show that the regulatory hierarchy among spadetail, no tail and a third T-box gene, tbx6, are substantially different during trunk and tail mesoderm formation, and propose a genetic model that accounts for the regional phenotypes of spadetail and no tail mutants.

The bZIP transcription factor LCR-F1 is essential for mesoderm formation in mouse development.

Genes Dev. 1997; 11: 786-98

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LCR-F1 is a mammalian bZIP transcription factor containing a basic amino acid domain highly homologous to a domain in the Drosophila Cap 'N' Collar and Caenorhabditis elegans SKN-1 proteins. LCR-F1 binds to AP1-like sequences in the human beta-globin locus control region and activates high-level expression of beta-globin genes. To assess the role of LCR-F1 in mammalian development, the mouse Lcrf1 gene was deleted in embryonic stem (ES) cells, and mice derived from these cells were mated to produce Lcrf1 null animals. Homozygous mutant embryos progressed normally to the late egg cylinder stage at approximately 6.5 days post coitus (dpc), but development was arrested before 7.5 dpc. Lcrf1 mutant embryos failed to form a primitive streak and lacked detectable mesoderm. These results demonstrate that LCR-F1 is essential for gastrulation in the mouse and suggest that this transcription factor controls expression of genes critical for the earliest events in mesoderm formation. Interestingly, Lcrf1 null ES cells injected into wild-type blastocysts contributed to all mesodermally derived tissues examined, including erythroid cells producing hemoglobin. These results demonstrate that the Lcrf1 mutation is not cell autonomous and suggest that LCR-F1 regulates expression of signaling molecules essential for gastrulation. The synthesis of normal hemoglobin levels in erythroid cells of chimeras derived from Lcrf1 null cells suggests that LCR-F1 is not essential for globin gene expression. LCR-F1 and the related bZIP transcription factors NF-E2 p45 and NRF2 must compensate for each other in globin gene regulation.

Gli family members are differentially expressed during the mitotic phase of spermatogenesis.

Oncogene. 1997; 14: 2259-64

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The Gli family of DNA binding proteins has been implicated in multiple neoplasias and developmental abnormalities, suggesting a primary involvement in cell development and differentiation. However, to date their specific roles and mechanisms of action remain obscure, and a drawback has been the lack of a model system in which to study their normal function. Here we demonstrate that Gli family members are differentially expressed during spermatogenesis in mice. Specifically, Gli and Gli3 mRNAs were detected in mouse germ cells, while Gli2 was not. Further, both Gli and Gli3 exhibited stage-dependent patterns of expression selectively in type A and B spermatogonia. Gli expression was somewhat higher in type B spermatogonia while the abundance of Gli3 transcripts was similar in type A and B cells. Gel-shift analyses also demonstrated the enrichment of DNA binding activity specific for the Gli target sequence in spermatogonial cells. These results indicate a selective role for Gli and Gli3 during mitotic stages of male germ cell development. Spermatogenesis may thus provide a unique opportunity to identify downstream targets and explore the normal function of Gli family proteins.

From the glossiphoniid leech Helobdella robusta, we have cloned and determined the complete coding sequence of Hro-nos, a gene homologous to the nanos gene from Drosophila melanogaster. Developmental northern blots show that Hro-nos, like nanos, is a maternal transcript that decays rapidly during early development. A polyclonal antiserum raised against the HRO-NOS protein was used in developmental western blots and for immunostaining leech embryos of different developmental stages. The HRO-NOS protein is first detectable in 2-cell embryos (4-6 hours of development) and exhibits a transient expression peaking during fourth cleavage (9-12 cells; 8-14 hours of development). The HRO-NOS protein exhibits a graded distribution along the primary embryonic axis and is partitioned unequally between the sister cells DNOPQ and DM, progeny of macromere D' at fourth cleavage: DNOPQ is the segmental ectoderm precursor cell and exhibits levels of HRO-NOS protein that are at least two-fold higher than in cell DM, the segmental mesoderm precursor cell. The observed expression pattern suggests that Hro-nos plays a role in the decision between ectodermal and mesodermal cell fates in leech.

tbx6, a Brachyury-related gene expressed by ventral mesendodermal precursors in the zebrafish embryo.

Dev Biol. 1997; 183: 61-73

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Classical embryology experiments have indicated the existence of dorsal-type and ventral-type mesoderms that arise as a consequence of mesoderm induction during vertebrate development. Here we report that the zebrafish tbx6 gene, a member of the Brachyury-related T-box family of genes, is exclusively expressed by ventral mesendoderm. Three observations link the expression of tbx6 to ventral mesoderm specification. First, the gene is initially expressed at the onset of gastrulation within a ventrolateral subpopulation of cells that express the pan-mesodermal gene, no tail (Brachyury). Second, the mesoderm-inducing factors activin and bFGF activate tbx6 expression in animal caps. Third, dorsalization of the mesendodermal precursor population following exposure of embryos to lithium ions causes down-regulation of tbx6 transcription. tbx6 is expressed transiently in the involuting derivatives of the ventral mesendoderm, which give rise to nonaxial mesodermal tissues; its expression is extinguished as tissue differentiation progresses. Transcription of tbx6 commences about an hour after initiation of expression of the pan-mesendodermal gene no tail and the organizer gene goosecoid. The dependence of tbx6 expression on no tail activity was examined in no tail mutant embryos. The activation of tbx6 transcription in ventral mesoderm does not depend on no tail gene activity. However, no tail appears to contribute to the maintenance of normal levels of tbx6 transcription and may be required for tbx6 transcription in the developing tail.

The identification of the mammalian testis-determining factor, SRY, led to the description of a new class of genes encoding transcription factors, the SOX gene family. SOX proteins display properties of both classical transcription factors and architectural components of chromatin. The dynamic and diverse patterns of expression of SOX genes and analysis of mutations in humans, mice and Drosophila suggest that SOX factors play key roles in decisions of cell fate during diverse developmental processes.

Brachyury, or T, is the founder member of a family of transcription factors that share the so-called T-box-a 200 amino acid DNA-binding domain. Recent work has addressed the regulation of Brachyury expression and its function in the embryo. New T-box family members have been found in vertebrate and invertebrate embryos and the importance of this gene family is illustrated by the discovery that mutations in human TBX5 are responsible for Holt-Oram syndrome, which is characterised by abnormalities in heart and forelimb development.

The mouse Brachyury (T) gene plays critical roles in the genesis of normal mesoderm during gastrulation and in the maintenance of a functioning notochord. Abrogation of Brachyury (T) expression within the chordamesoderm of homozygous null mutants nevertheless spares anterior axis formation. An intriguing possibility to explain the preservation of anterior axis formation in these mutants would be the existence of other genes compensating for the loss of Brachyury. This compensation and the recent demonstration that Brachyury is the prototype for an evolutionarily conserved family, prompted a search for other T-box genes participating in axis formation. The chick Brachyury orthologue and two related chick T-box genes that are expressed at the onset of gastrulation have been isolated. One of these novel genes (Ch-TbxT) becomes restricted to the axial mesoderm lineage and is a potential candidate for complementing or extending Brachyury function in the anterior axis (formation of the head process, prechordal plate). The other gene (Ch-Tbx6L), together with chick T, appears to mark primitive streak progenitors before gastrulation. As cells leave the primitive streak, Ch-Tbx6L becomes restricted to the early paraxial mesoderm lineage and could play a role in regulating somitogenesis.

The Pax gene family consists of tissue-specific transcriptional regulators that always contain a highly conserved DNA-binding domain with six alpha-helices (paired domain), and, in many cases, a complete or residual homeodomain. Numerous genes of this family have been identified in animals, with the largest number found in vertebrates. Our evolutionary analyses indicate that the vertebrate Pax gene family consists of four well-defined and statistically supported groups: group I (Pax-1, 9), II (Pax-2, 5, 8), III (Pax-3, 7), and IV (Pax-4, 6). Group I paired domains share a most recent common ancestor with Drosophila Pox meso, group II with Pox neuro, group III with paired and gooseberry, and group IV with the eyeless gene. Two groups containing complete homeodomains (III and IV) are distantly related, and the intergroup relationships are (I,III), (II,IV). These four major groups arose before the divergence of Drosophila and vertebrates prior to the Cambrian radiation of triploblastic metazoan body plans. We conducted an analysis of fixed radical amino acid differences between groups in a phylogenetic context. We found that all four fixed radical amino acid differences between groups I and III are located exclusively in the N-terminal alpha-helices. Similarly, groups II and IV show three fixed radical differences in these alpha-helices but at positions different from those in groups I and III. Implications of such fixed amino acid differences in potentially generating sequence recognition specificities are discussed in the context of some recent experimental findings.

The T-box gene family consists of members that share a unique DNA binding domain. The best characterized T-box gene, Brachyury or T, encodes a transcription factor that plays an important role in early vertebrate development. Seven other recently described mouse T-box genes are also expressed during development. In the nematode Caenorhabditis elegans, four T-box genes have been characterized to date. In this study, we describe three new C. elegans T-box genes, named Ce-tbx-11, Ce-tbx-12, and Ce-tbx-17. Ce-tbx-11 and Ce-tbx-17 were uncovered through the sequencing efforts of the C. elegans Genome Project. Ce-tbx-12 was uncovered through degenerate PCR analysis of C. elegans genomic DNA. Ce-tbx-11 and Ce-tbx-17 are located in close proximity to the four other previously described T-box genes in the central region of chromosome III. In contrast, Ce-tbx-12 maps alone to chromosome II. Phylogenetic analysis of all known T-box domain sequences provides evidence of an ancient origin for this gene family.

Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome.

Nat Genet. 1997; 16: 311-5

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Ulnar-mammary syndrome is a rare pleiotropic disorder affecting limb, apocrine gland, tooth and genital development. We demonstrate that mutations in human TBX3, a member of the T-box gene family, cause ulnar-mammary syndrome in two families. Each mutation (a single nucleotide deletion and a splice-site mutation) is predicted to cause haploinsufficiency of TBX3, implying that critical levels of this transcription factor are required for morphogenesis of several organs. Limb abnormalities of ulnar-mammary syndrome involve posterior elements. Mutations in TBX5, a related and linked gene, cause anterior limb abnormalities in Holt-Oram syndrome. We suggest that during the evolution of TBX3 and TBX5 from a common ancestral gene, each has acquired specific yet complementary roles in patterning the mammalian upper limb.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.

Nat Genet. 1997; 15: 21-9

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Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago. We have now identified a gene for this disorder (HOS1). The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse Tbx5 gene. We have identified six mutations, three in HOS families and three in sporadic HOS cases. Each of the mutations introduces a premature stop codon in the TBX5 gene product. Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBX5 in heart and limb, consistent with a role in human embryonic development.

The T-box genes constitute an evolutionarily conserved family of putative transcription factors which are expressed in discrete domains during embryogenesis, suggesting that they may play roles in inductive interactions. Members have been identified by virtue of their homology to the prototypical T-box gene, T or Brachyury, which is required for mesoderm formation and axial elongation during embryogenesis. We have previously reported the discovery of six new mouse T-box genes, Tbx1-Tbx6, and described the expression patterns of Tbx1-Tbx5 (Bollag et al., 1994; Agulnik et al., 1996; Chapman et al., 1996; Gibson-Brown et al., 1996). We have obtained cDNA clones encoding the full-length Tbx6 protein from screens of gastrulation-stage mouse cDNA libraries and determined the spatial and temporal distribution of Tbx6 transcripts during embryogenesis. The gene codes for a 1.9-kb transcript with an open reading frame coding for a 540-amino acid protein, with a predicted molecular weight of 59 kDa. Tbx6 maps to chromosome 7 and does not appear to be linked to any known mutation. Unlike other members of the mouse T-box gene family which are expressed in a wide variety of tissues derived from all germ layers, Tbx6 expression is quite restricted. Tbx6 transcripts are first detected in the gastrulation stage embryo in the primitive streak and newly recruited paraxial mesoderm. Later in development, Tbx6 expression is restricted to presomitic, paraxial mesoderm and to the tail bud, which replaces the streak as the source of mesoderm. Expression in the tail bud persists until 12. 5 days postcoitus. Tbx6 expression thus overlaps that of Brachyury in the primitive streak and tail bud, although Brachyury is expressed earlier in the primitive streak. Brachyury is also expressed in a second domain, the node and notochord, that is not shared with Tbx6. The onset of Tbx6 expression is not affected in homozygous null Brachyury mutant embryos at 7.5 days postcoitus. However, Tbx6 expression is extinguished in mutant embryos as soon as the Brachyury phenotype becomes evident at 8.5 days postcoitus, indicating that the continued expression of Tbx6 is directly or indirectly dependent upon Brachyury expression.

Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development.

Dev Dyn. 1996; 206: 379-90

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A novel family of genes, characterized by the presence of a region of homology to the DNA-binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T-box, and the new mouse genes that contain the T-box domain have been named T-box 1-6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1-Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3-5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2-5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T-box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.

Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity.

Mech Dev. 1996; 56: 93-101

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Tetrapod fore-and hindlimbs have evolved from the pectoral and pelvic fins of an ancient vertebrate ancestor. In this ancestor, the pectoral fin appears to have arisen following the rostral homeotic recapitulation of an existing pelvic appendage (Tabin and Laufer (1993), Nature 361, 692-693). Thus the basic appendage outgrowth program is reiterated in both tetrapod fore- and hindlimbs and the pectoral and pelvic fins of extant teleost fishes (Sordino et al. (1995) Nature 375, 678-681). Recently a novel family of putative transcription factors, which includes the T (Brachyury) locus, has been identified and dubbed the "T-box' family. In mice, all of these genes have expression patterns indicative of involvement in embryonic induction (Chapman et al. (1996) Dev. Dyn., in press), and four (Tbx2-Tbx5) are represented as two cognate, linked gene pairs (Agulnik et al., (1996), Genetics, in press). We now report that, whereas Tbx2 and Tbx3 are expressed in similar spatiotemporal patterns in both limbs, Tbx5 and Tbx4 expression is primarily restricted to the developing fore- and hindlimb buds, respectively. These observations suggest that T-box genes have played a role in the evolution of fin and limb morphogenesis, and that Tbx5 and Tbx4 may have been divergently selected to play a role in the differential specification of fore- (pectoral) versus hind- (pelvic) limb (fin) identity.

The T-box genes comprise an ancient family of putative transcription factors conserved across species as divergent as Mus musculus and Caenorhabditis elegans. All T-box gene products are characterized by a novel 174-186-amino acid DNA binding domain called the T-box that was first discovered in the polypeptide products of the mouse T locus and the Drosophila melanogaster optomotor-blind gene. Earlier studies allowed the identification of five mouse T-box genes, T, Tbx1-3, and Tbr1, that all map to different chromosomal locations and are expressed in unique temporal and spatial patterns during embryogenesis. Here, we report the discovery of three new members of the mouse T-box gene family, named Tbx4, Tbx5, and Tbx6. Two of these newly discovered genes, Tbx4 and Tbx5, were found to be tightly linked to previously identified T-box genes. Combined results from phylogenetic, linkage, and physical mapping studies provide a picture for the evolution of a T-box subfamily by unequal crossing over to form a two-gene cluster that was duplicated and dispersed to two chromosomal locations. This analysis suggests that Tbx4 and Tbx5 are cognate genes that diverged apart from a common ancestral gene during early vertebrate evolution.

Rescue of the hairless phenotype in nude mice by transgenic insertion of the wild-type Hfh11 genomic locus.

Int Immunol. 1996; 8: 961-6

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Mice and rats homozygous for mutations at the nude (nu) locus exhibit the pleiotropic phenotypes of hairlessness and athymia. A recent positional cloning study identified, as a nude gene, a novel fork head transcription factor, Hfh11 (also called whn), that is expressed in skin and thymus, and is mutated in nude rodents. To obtain the direct biological proof that this gene is responsible for nude phenotype, we microinjected a cosmid clone containing the wild-type Hfh11 genomic locus into fertilized nude eggs. Two independent founder lines of transgenic mice were generated that corrected the hairless phenotype, but not the thymic defect. This partial rescue demonstrates that Hfh11 is the gene responsible for the hairless defect in the nude mouse. Taken together with previous genetic studies, this complementation result indicates that Hfh11 is indeed the nude gene and the Hfh11 locus is likely to be subject to complicated regulation.

A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.

Mol Cell Biol. 1996; 16: 7133-43

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RecA in Escherichia coli and its homolog, ScRad51 in Saccharomyces cerevisiae, are known to be essential for recombinational repair. The homolog of RecA and ScRad51 in mice, MmRad51, was mutated to determine its function. Mutant embryos arrested early during development. A decrease in cell proliferation, followed by programmed cell death and chromosome loss, was observed. Radiation sensitivity was demonstrated in trophectoderm-derived cells. Interestingly, embryonic development progressed further in a p53 null background; however, fibroblasts derived from double-mutant embryos failed to proliferate in tissue culture.

We have used differential display to identify genes inducible by activin and isolated a novel member of the T-box gene family that includes the Xenopus genes Xbrachyury and Eomesodermin. Here we show that this novel gene is unique within the T-box family because it is maternally expressed at a high level. Furthermore, it belongs to a rare class of maternal mRNAs in Xenopus that are localised to the vegetal hemisphere of the egg and we have therefore named it Antipodean. We show here that low amounts of Antipodean injected into ectoderm (animal cap cells) strongly induce pan mesodermal genes such as Xbrachyury and ventral mesodermal genes such as Xwnt-8. Overexpression of Antipodean generates mesoderm of ventral character, and induces muscle only weakly. This property is consistent with the observed late zygotic Antipodean mRNA expression in the posterior paraxial mesoderm and ventral blastopore, and its exclusion from the most dorsal mesodermal structure, the notochord. Antipodean is induced by several molecules of the TGF-beta class, but in contrast to Xbrachyury, not by bFGF. This result suggests that the expression of these T-box genes may be under the control of different regulatory pathways. Finally, we demonstrate that Antipodean and Eomesodermin induce each other and both are able to induce Xbrachyury. The early zygotic expression of Antipodean is not induced by Xbrachyury, though later it is to some extent. Considering its maternal content, Antipodean could initiate a cascade of T-box gene activations. The expression of these genes may, in turn, sustain each other's expression to define and maintain the mesoderm identity in Xenopus.

A fork head related multigene family is transcribed in Xenopus laevis embryos.

Int J Dev Biol. 1996; 40: 245-53

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We have isolated and sequenced ten different members of the fork head/HNF-3 multigene family from Xenopus laevis which have been termed Xenopus fork head domain related (XFD) genes 1 to 10. Another four isolated genes (XFD' genes) represent pseudo-allelic variants which arose by an ancient tetraploidization within this species. Whereas all genes of this multigene family exhibit a high degree of sequence homology within the evolutionary conserved fork head domain, sequences outside this module are substantially different. Based upon sequence homologies over the entire coding sequences, XFD-7/7' represent the Xenopus homologs to the rodent hepatocyte nuclear factor HNF-3 alpha, while XFD-3/3' encode the homologs to HNF-3 beta. Here we present an analysis of the temporal transcription pattern of XFD genes 1 to 10 during embryogenesis and in some adult tissues. Eight of these XFD genes are activated during embryonic development, but show different and distinct transcription profiles. The localization of transcripts was determined by whole-mount in situ hybridization. Although transcription of individual XFD genes partially overlaps, each gene is characterized by means of a specific spatial pattern of transcriptional activity.

Evolutionary strategies for the elucidation of cis and trans factors that regulate the developmental switching programs of the beta-like globin genes.

Mol Phylogenet Evol. 1996; 5: 18-32

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We describe three strategies for the identification of specific cis and trans factors that regulate globin gene expression, all three of which are based on the evolution of the globin genes and their expression patterns. The first approach, phylogenetic footprinting, relies on a search for sequence similarities and is designed to elucidate the factors that control those expression patterns which are shared by orthologous globin genes of all eutherian mammals (e.g., the expression of the epsilon globin genes in the embryonic yolk sac and its repression in fetal and adult hematopoietic tissues). The second approach, differential phylogenetic footprinting, relies on a search for sequence differences. This approach may be of value in identifying the mechanisms underlying the generation of novel expression patterns in specific lineages (e.g., the expression of gamma as a fetal gene in the simian primates in contrast with the embryonic expression of gamma in all other mammals). Finally, motif-based phylogenetic analysis takes into consideration the fact that many transcription factors are quite flexible in the recognition of their cognate sites. The approach allows the detection of functionally conserved binding sites despite their sequence variation.

Role of notochord in specification of cardiac left-right orientation in zebrafish and Xenopus.

Dev Biol. 1996; 177: 96-103

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The left-right body axis is coordinately aligned with the orthogonal dorsoventral and anterioposterior body axes. The developmental mechanisms that regulate axis coordination are unknown. Here it is shown that the cardiac left-right orientation in zebrafish (Danio rerio) is randomized in notochord-defective no tail and floating head mutants. no tail (Brachyury) and floating head (Xnot) encode putative transcription factors that are expressed in the organizer and notochord, structures which regulate dorsoventral and anterioposterior development in vertebrate embryos. Results from dorsal tissue extirpation and cardiac primordia explantation indicate that cardiac left-right orientation is dependent on dorsoanterior structures including the notochord and is specified during neural fold stages in Xenopus laevis. Thus, the notochord coordinates the development of all three body axes in the vertebrate body plan.

The developmental regulation of the human beta-globin cluster embodies all aspects of transcriptional control of eukaryotic genes. The cis-acting sequences within the cluster, distal regulatory regions and trans-acting factors all contribute to provide stringent temporal and tissue-specific expression. This review will examine the individual regulatory mechanisms which govern globin gene expression and highlight recent advances which expand our understanding of these dynamic interactions.

Expression of the E2F-1/DP-1 transcription factor in murine development.

Cell Growth Differ. 1996; 7: 43-52

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Because of its critical role in the control of cell proliferation and differentiation, we postulated that E2F-1 could have a role in murine development. To this end, the organ and developmental expression of the E2F-1 transcription factor was analyzed from mid-gestation to late-stage embryogenesis. We demonstrate that the mRNA levels for E2F-1 and its heterodimeric partner DP-1 reach maximal levels in the late embryonic and early postnatal period but decline in the later postnatal and adult periods. Additionally, using high resolution in situ hybridization, high expression of E2F-1 was observed in specific cells of individual tissues, suggesting that the role of E2F-1 may be more complex than previously indicated from cell culture studies. Furthermore, the unusual pattern of E2F-1 and DP-1 developmental expression may have an essential role in certain cells and tissues in the late embryonic and early postnatal period.

The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo.

Dev Biol. 1996; 180: 773-9

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The vertebrate Brachyury (T) gene is transiently expressed in nascent and migrating mesoderm, in the differentiating notochord, and in the tail bud, reflecting its independent functions. In contrast, the expression of an ascidian Brachyury gene (As-T) is restricted to differentiating notochord. The present study revealed that the genome of Halocynthia roretzi contains another T-domain gene (As-T2) which encodes a divergent T-domain protein. The transient expression of As-T2 was detected in the endoderm- and muscle-lineage blastomeres of the early embryo and the transcript was retained by involuting and differentiating muscle cells until it became undetectable by the mid-tailbud stage. In addition, As-T2 was expressed transiently in cells that form the tip of the newly forming tail. Interestingly, the combined pattern of spatial expression of As-T and As-T2 appears to correspond to that of a single vertebrate Brachyury gene.

The hepatocyte nuclear factor-3/forkhead transcription regulatory family in development, inflammation, and neoplasia.

Crit Rev Oncol Hematol. 1995; 20: 129-40

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HNF-3/FKH genes are a large family of transcriptional activators. They are expressed in specific developmental and tissue patterns. Indeed, several of them are known to be essential for normal development (e.g. Dfkh and slp-1,2). Mutation within one of these genes produces mutant fruitfly embryos that are unable to survive. This family shares conserved DNA binding and transcriptional activation domains. The DNA binding domain has been crystallized, and its structure determined. Although it has resemblance to helices of homeodomains and H5 histones, it represents a new DNA binding motif, which has been called the 'winged helix,' because it contains additional interactive peptide regions called termed wings. Subtle amino acid variations in a region adjacent to the DNA recognition helix influence the recognition specificity of each HNF-3/FKH protein and therefore confer selectivity in promoter regulation. Members of this family are important in regulating the inflammatory response of the liver (the three HNF-3 genes). In addition, several members may be important in blood cell development (H3 and 5-3). Finally, two of these genes have been found to produce neoplasia (qin and FKHR). As investigation progresses, the mechanism by which these genes regulate development, inflammation and neoplasia will become more clear.

Chasing tails in ascidians: developmental insights into the origin and evolution of chordates.

Trends Genet. 1995; 11: 354-9

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The ascidian tadpole larva is regarded as a prototype of the ancestral chordate. Here we consider recent studies on the development of the tadpole larva that provide new insights into chordate origins and evolution. The notochord of ascidian larvae and vertebrates appear to be homologous structures based on their induction by endoderm and expression of the Brachyury (T) gene. The muscle cells of ascidian larvae also appear homologous to those of vertebrates based on their expression of bHLH myogenic and muscle-type actin genes, although they are specified by cytoplasmic determinants localized in the egg as well as embryonic induction. Studies of the tailless larvae of anural ascidians have resulted in the identification of Manx, a gene that may control tail development and evolution. These and other results support the ascidian tadpole prototype for the ancestral chordate.

The Drosophila runt locus controls early events in embryogenesis. A human homologue (CBFA2) was originally identified because of its involvement in the t(8;21) associated with a subtype of acute myeloid leukaemia. The phylogenetically conserved region (runt box) was reported to correspond to a DNA binding domain. In order to investigate whether runt also plays a role in mammalian development, we have conducted a preliminary survey of its expression in the mouse embryo. Expression in embryonic tissues was detected starting from day 9.2 post coitum. From day 10.5 post coitum, highest levels are found in the neural tube, sensory ganglia, specialised sensory epithelial structures (olfactory and gustatory mucosa, follicles of the vibrissae), all chondrogenic centres (both of neural crest and of mesodermal origin), and the genital system (the gonad, the paramesonephros, and the genital tubercle). Unambiguous expression in the haemopoietic system could be established for the thymus. The data suggest a pleiotropic role for mammalian runt in embryogenesis.

Conservation of the T-box gene family from Mus musculus to Caenorhabditis elegans.

Genomics. 1995; 25: 214-9

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Recently, a novel family of genes with a region of homology to the mouse T locus, which is known to play a crucial, and conserved, role in vertebrate development, has been discovered. The region of homology has been named the T-box. The T-box domain of the prototypical T locus product is associated with sequence-specific DNA binding activity. In this report, we have characterized four members of the T-box gene family from the nematode Caenorhabditis elegans. All lie in close proximity to each other in the middle of chromosome III. Homology analysis among all completely sequenced T-box products indicates a larger size for the conserved T-box domain (166 to 203 residues) than previously reported. Phylogenetic analysis suggests that one C. elegans T-box gene may be a direct ortholog of the mouse Tbx2 and Drosophila omb genes. The accumulated data demonstrate the ancient nature of the T-box gene family and suggest the existence of at least three separate T-box-containing genes in a common early metazoan ancestor to nematodes and vertebrates.

The mouse Brachyury gene (T) is required in notochord differentiation and posterior mesoderm formation during axial development. We have isolated the chick homologue of T(Ch-T) and determined its putative protein sequence and expression pattern during embryogenesis. Ch-T is expressed in the epiblast close to and within the primitive streak, in early migrating mesoderm and in the notochord. In later stages Ch-T expression is found in the tail bud and in the entire notochord. The notochord expression ceases in an anterior-posterior wave when the formation of the body anlage is completed. This pattern is consistent with those reported for the expression of the mouse T gene and the T homologues of Xenopus laevis and zebrafish, suggesting that the mechanisms of embryonic pattern formation are highly conserved in all vertebrates. The N-terminal half of Ch-T shows a very high degree of sequence identity with the corresponding region of mouse T which has DNA-binding activity, and with the N-terminal half of Xenopus (Xbra) and zebrafish (Ntl) T protein. Finally, we have analyzed the effects of activin A on Ch-T induction and axis formation. Localized activin A treatment of prestreak blastoderms results in ectopic Ch-T expression that correlates with formation of second primitive streaks or with repositioning of the site of single streak origin (Cooke et al., 1994). These results strengthen the previous evidence that Brachyury activation is an early response to axis-inducing signals in vivo.

In the broadest terms, epigenetic phenomena in eukaryotes depend on the interaction of alleles or repeated sequences or on the mitotic inheritance of chromatin states or methylation patterns. One of the most exciting aspects of the study of epigenetic phenomena is the insight that can be gained into the structure and assembly of higher-order chromatin structures, an important subject that has proved refractory to current biochemical methodologies. Rapid progress in the study of gene inactivation in fungi, plants, and invertebrates will provide new hypotheses to be tested in mammals.

Since its identification in 1927, the mouse T (Brachyury) locus has been implicated in mesoderm formation and notochord differentiation. Recent work has demonstrated that this gene encodes a putative transcription factor expressed specifically in nascent mesoderm and in the differentiating notochord. Homologous genes have been cloned from the frog Xenopus laevis, the zebrafish Brachydanio rerio and the ascidian Halocynthia roretzi. The T gene is an important tool for elucidating mesoderman and embryonic pattern formation.

Brachyury (T) mutant embryos are deficient in mesoderm formation and do not complete axial development. The notochord is most strongly affected. The T gene is expressed transiently in primitive streak-derived nascent and migrating mesoderm cells and continuously in the notochord. Ectopic expression of T protein in the animal cap of Xenopus embryos results in ectopic mesoderm formation. The T protein is located in the nucleus. These and other data suggested that the T gene might be involved in the control of transcriptional regulation. In an attempt to demonstrate specific DNA binding of the T protein we have identified a consensus sequence among DNA fragments selected from a mixture of random oligomers. Under our experimental conditions T protein binds as a monomer to DNA. This property resides in the N-terminal domain of 229 amino acid residues which is strongly conserved between the mouse protein, and its Xenopus and zebrafish homologues. The latter proteins also recognize the consensus DNA binding site. We suggest that the T protein is involved in the control of genes required for mesoderm formation, and for the differentiation and function of chorda mesoderm.